Trichloroethylene-induced alterations in DNA methylation were enriched in polycomb protein binding sites in effector/memory CD4+ T cells

Abstract

Exposure to industrial solvent and water pollutant trichloroethylene (TCE) can promote autoimmunity, and expand effector/memory (CD62L) CD4⁺ T cells. In order to better understand etiology reduced representation bisulfite sequencing was used to study how a 40-week exposure to TCE in drinking water altered methylation of ∼337 770 CpG sites across the entire genome of effector/memory CD4⁺ T cells from MRL+/+ mice. Regardless of TCE exposure, 62% of CpG sites in autosomal chromosomes were hypomethylated (0-15% methylation), and 25% were hypermethylated (85-100% methylation). In contrast, only 6% of the CpGs on the X chromosome were hypomethylated, and 51% had mid-range methylation levels. In terms of TCE impact, TCE altered (≥ 10%) the methylation of 233 CpG sites in effector/memory CD4⁺ T cells. Approximately 31.7% of these differentially methylated sites occurred in regions known to bind one or more Polycomb group (PcG) proteins, namely Ezh2, Suz12, Mtf2 or Jarid2. In comparison, only 23.3% of CpG sites not differentially methylated by TCE were found in PcG protein binding regions. Transcriptomics revealed that TCE altered the expression of ∼560 genes in the same effector/memory CD4⁺ T cells. At least 80% of the immune genes altered by TCE had binding sites for PcG proteins flanking their transcription start site, or were regulated by other transcription factors that were in turn ordered by PcG proteins at their own transcription start site. Thus, PcG proteins, and the differential methylation of their binding sites, may represent a new mechanism by which TCE could alter the function of effector/memory CD4⁺ T cells.

Keywords: DNA methylation; autoimmunity; immunotoxicity; polycomb proteins; trichloroethylene.

Figure 1:

Average DNA methylation levels of all CpGs interrogated. RRBS analysis of the effector/memory CD4⁺ T cells collected after 40 weeks of adult exposure to TCE was conducted. (A) Histograms show the average methylation of all 337 770 CpG sites examined in CD4⁺ T cells from either control or TCE-treated mice after binning for average methylation (e.g. 0–5% methylation or 20–25% methylation). (B) The same histograms are shown without inclusion of the CpGs that were either 0–5% or 95–100% methylated

Figure 2:

Chromosome-specific mean DNA methylation levels. (A) The results from the RRBS analysis described in Fig. 1 were sorted into individual chromosomes, and presented after binning for average methylation of the CpGs. The area of each histogram was normalized to one to make it easier to compare the chromosomes. (B) The RRBS results were presented (as total number of CpG sites in the different bins) after excluding the CpG sites that were either 0–5% or 95–100% methylated

Figure 3:

TCE exposure decreased total methylation variance. The RRBS results for the effector/memory CD4⁺ T cells from control or TCE-treated mice were sorted by treatment group and binned for mean methylation. The inter-sample methylation variance at all the CpG sites in the different bins was then calculated. The dotted line represents a prediction of highest possible methylation variance based on a theoretical value spread of four samples in each bin

Figure 4:

Identification of CpG sites differentially methylated by TCE exposure. (A) RRBS analysis of effector/memory CD4⁺ T cells from control and TCE-treated mice revealed 233 DMS (methylation difference ≥ 10%, q value <0.005). Hierarchical clustering of the gene-associated DMS is shown here. (B) The genomic location of the 233 DMS detected in the effector/memory CD4⁺ T cells relative to the nearest transcription start site (TSS) is shown. (C) The genes associated with the DMS identified by the RRBS were subjected to a gene list functional analysis by the Panther Gene Ontology Classification System

Figure 5:

Average percent methylation of CpG sites differentially methylated by TCE. RRBS analysis of effector/memory CD4⁺ T cells from control and TCE-treated mice revealed 233 DMS (methylation difference ≥ 10%, q value < 0.005). These DMS were sorted separately for control and TCE-treated samples and binned for mean methylation

Figure 6:

Many CpG sites differentially methylated by TCE are found in PcG protein binding sites. RRBS analysis of effector/memory CD4⁺ T cells from control and TCE-treated mice revealed 233 DMS (methylation difference ≥ 10%, q value < 0.005). Annotation of these DMS described whether they were found outside of a transcription binding site (NRE: no regulatory element), or in a regulatory element that bound PcG proteins or other transcription factors. The percentage of CpG sites differentially methylated by TCE was compared with 400 randomly selected CpG sites not altered by TCE (Random CpGs), and to the total number of individual OREG sites known to bind Suz12, EZH2, Mtf2 or Jarid2 or other transcription factors (as identified in Mouse NCB137/mm9 genome in the UCSC Genome Browser) (All Regulatory Elements in Genome)